ABSTRACT
Recombinant mammalian host cell lines, in particular CHO and HEK293 cells, are used for the industrial production of therapeutic proteins. Despite their well-known genomic instability, the control mechanisms that enable cells to respond to changes in the environmental conditions are not yet fully understood, nor do we have a good understanding of the factors that lead to phenotypic shifts in long-term cultures. A contributing factor could be inherent diversity in transcriptomes within a population. In this study, we used a full-length coverage single-cell RNA sequencing (scRNA-seq) approach to investigate and compare cell-to-cell variability and the impact of standardized and homogenous culture conditions on the diversity of individual cell transcriptomes, comparing suspension CHO-K1 and adherent HEK293FT cells. Our data showed a critical batch effect from the sequencing of four 96-well plates of CHO-K1 single cells stored for different periods of time, which was and may be therefore identified as a technical variable to consider in experimental planning. Besides, in an artificial and controlled culture environment such as used in routine cell culture technology, the gene expression pattern of a given population does not reveal any marker gene capable to disclose relevant cell population substructures, both for CHO-K1 cells and for HEK293FT cells. The variation observed is primarily driven by the cell cycle.
Subject(s)
Transcriptome , Cricetinae , Animals , Humans , Transcriptome/genetics , HEK293 Cells , CHO Cells , Cricetulus , Sequence Analysis, RNAABSTRACT
As of today, the majority of environmental microorganisms remain uncultured. They are therefore referred to as "microbial dark matter." In the recent past, cultivation-independent methods like single-cell genomics (SCG) enabled the discovery of many previously unknown microorganisms, among them the Patescibacteria/Candidate Phyla Radiation (CPR). This approach was shown to be complementary to metagenomics, however, the development of additional and refined sorting techniques beyond the most commonly used fluorescence-activated cell sorting (FACS) is still desirable to enable additional downstream applications. Adding image information on the number and morphology of sorted cells would be beneficial, as would be minimizing cell stress caused by sorting conditions such as staining or pressure. Recently, a novel cell sorting technique has been developed, a microfluidic single-cell dispenser, which assesses the number and morphology of the cell in each droplet by automated light microscopic processing. Here, we report for the first time the successful application of the newly developed single-cell dispensing system for label-free isolation of individual bacteria from a complex sample retrieved from a wastewater treatment plant, demonstrating the potential of this technique for single cell genomics and other alternative downstream applications. Genome recovery success rated above 80% with this technique-out of 880 sorted cells 717 were successfully amplified. For 50.1% of these, analysis of the 16S rRNA gene was feasible and led to the sequencing of 50 sorted cells identified as Patescibacteria/CPR members. Subsequentially, 27 single amplified genomes (SAGs) of 15 novel and distinct Patescibacteria/CPR members, representing yet unseen species, genera and families could be captured and reconstructed. This phylogenetic distinctness of the recovered SAGs from available metagenome-assembled genomes (MAGs) is accompanied by the finding that these lineages-in whole or in part-have not been accessed by genome-resolved metagenomics of the same sample, thereby emphasizing the importance and opportunities of SCGs.
ABSTRACT
Single-cell multi-omics are powerful means to study cell-to-cell heterogeneity. Here, we present a single-tube, bisulfite-free method for the simultaneous, genome-wide analysis of DNA methylation and genetic variants in single cells: epigenomics and genomics of single cells analyzed by restriction (epi-gSCAR). By applying this method, we obtained DNA methylation measurements of up to 506,063 CpGs and up to 1,244,188 single-nucleotide variants from single acute myeloid leukemia-derived cells. We demonstrate that epi-gSCAR generates accurate and reproducible measurements of DNA methylation and allows to differentiate between cell lines based on the DNA methylation and genetic profiles.
Subject(s)
DNA Methylation , Epigenesis, Genetic , Epigenome , Epigenomics , Leukemia, Myeloid, Acute/genetics , Single-Cell Analysis , Cell Line, Tumor , CpG Islands , Gene Expression Regulation, Neoplastic , High-Throughput Nucleotide Sequencing , Humans , Polymerase Chain Reaction , Polymorphism, Single Nucleotide , RNA-Seq , Reproducibility of ResultsSubject(s)
Blast Crisis , Fusion Proteins, bcr-abl , Leukemia, Myelogenous, Chronic, BCR-ABL Positive , Oncogene Proteins, Fusion , Proto-Oncogene Proteins c-kit , Pyrimidines/administration & dosage , Adult , Blast Crisis/drug therapy , Blast Crisis/genetics , Blast Crisis/metabolism , Blast Crisis/pathology , Fusion Proteins, bcr-abl/genetics , Fusion Proteins, bcr-abl/metabolism , Humans , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Male , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , Proto-Oncogene Proteins c-kit/genetics , Proto-Oncogene Proteins c-kit/metabolismABSTRACT
Single-cell dispensing for automated cell isolation of individual cells has gained increased attention in the biopharmaceutical industry, mainly for production of clonal cell lines. Here, machine learning for classification of cell images is applied for 'real-time' cell viability sorting on a single-cell printer. We show that an extremely shallow convolutional neural network (CNN) for classification of low-complexity cell images outperforms more complex architectures. Datasets with hundreds of cell images from four different samples were used for training and validation of the CNNs. The clone recovery, i.e. the fraction of single-cells that grow to clonal colonies, is predicted to increase for all the samples investigated. Finally, a trained CNN was deployed on a c.sight single-cell printer for 'real-time' sorting of a CHO-K1 cells. On a sample with artificially damaged cells the clone recovery could be increased from 27% to 73%, thereby resulting in a significantly faster and more efficient cloning. Depending on the classification threshold, the frequency at which viable cells are dispensed could be increased by up to 65%. This technology for image-based cell sorting is highly versatile and can be expected to enable cell sorting by computer vision with respect to different criteria in the future.
Subject(s)
Cell Proliferation/physiology , Cell Survival/physiology , Clone Cells/metabolism , Machine Learning , Neural Networks, Computer , Animals , Antibodies, Monoclonal/therapeutic use , CHO Cells , Computer Simulation , Cricetulus , Image Cytometry , Recombinant Proteins/therapeutic useABSTRACT
We report a method that enables untargeted, high throughput, and quantitative mass spectrometric analysis of single cells from cell suspension without needing additional sample preparation procedures (e.g., molecular tagging) through the combination of single-cell printer technology and liquid vortex capture-mass spectrometry (SCP-LVC-MS). The operating principle behind the SCP-LVC-MS technology is single cell isolation via small droplet piezoelectric ejection followed by capture of the droplet into an LVC-MS sampling probe. Once exposed to an appropriate solvent, the cell is lysed, extracted, and analyzed by MS. The SCP-LVC-MS approach was validated by measuring the lipid composition of microalgae, Chlamydomonas reinhardtii (ChRe) and Euglena gracilis (EuGr), and HeLa cells in their native growth media. Numerous diacylglyceryltrimethylhomo-Ser (DGTS), phosphatidylcholine (PC), monogalactosyldiacylglycerol (MGDG), and digalactosyldiacylglycerol (DGDG) lipids were observed in single cells. Continuous solvent flow ensures that cells are analyzed rapidly, and no signal carryover between cells is observed. ChRe and EuGr microalgae mixed together in the same solution were differentiated cell-by-cell in real-time based on differences between levels of diacylglyceryltrimethylhomo-Ser (DGTS) and phosphatidylcholine (PC) lipids measured in each cell. Several DGTS lipids present in ChRe were quantified with single-cell resolution by normalizing to a DGTS(32:0) internal standard added to the LVC probe solvent during analysis. Quantitative peak areas were validated by comparing to bulk lipid extracts. Lastly, peak area distributions comprised of hundreds of cells were compared for ChRe after 5 days of nitrogen-limited and normal growth conditions, which show clear differences and the ability to resolve cellular population differences with single-cell resolution.
Subject(s)
Laser Capture Microdissection , Lipids/analysis , Single-Cell Analysis , Chlamydomonas reinhardtii/chemistry , Chlamydomonas reinhardtii/cytology , Chlamydomonas reinhardtii/growth & development , Euglena gracilis/chemistry , Euglena gracilis/cytology , Euglena gracilis/growth & development , HeLa Cells , Humans , Mass Spectrometry , Particle Size , Surface PropertiesABSTRACT
Knowledge of the molecular and clonal characteristics in the myelodysplastic syndromes (MDS) and during progression to acute myeloid leukaemia (AML) is essential to understand the disease dynamics and optimize treatment. Sequencing serial bone marrow samples of eight patients, we observed that MDS featured a median of 3 mutations. Mutations in genes involved in RNA-splicing or epigenetic regulation were most frequent, and exclusively present in the major clone. Minor subclones were distinguishable in three patients. As the MDS progressed, a median of one mutation was gained, leading to clonal outgrowth. No AML developed genetically independent of a pre-existing clone. The gained mutation mostly affected genes encoding signalling proteins. Additional acquisition of genomic aberrations frequently occurred. Upon treatment, emergence of new clones could be observed. As confirmed by single-cell sequencing, multiple mutations in identical genes in different clones were present within individual patients. DNA-methylation profiling in patients without identification of novel mutations in AML revealed methylation changes in individual genes. In conclusion, our data complement previous observations on the mutational and clonal characteristics in MDS and at progression. Moreover, DNA-methylation changes may be associated with progression in single patients. Redundancy of mutated genes in different clones suggests fertile grounds promoting clonal selection or acquisition.
Subject(s)
Clone Cells/pathology , Disease Progression , Leukemia, Myeloid, Acute/genetics , Myelodysplastic Syndromes/genetics , Myelodysplastic Syndromes/pathology , Adult , DNA Methylation , Female , Humans , Leukemia, Myeloid, Acute/etiology , Male , Middle Aged , Mutation , Myelodysplastic Syndromes/therapy , Single-Cell AnalysisABSTRACT
In tissue engineering applications, vascularization can be accomplished by coimplantation of tissue forming cells and endothelial cells (ECs), whereby the latter are able to form functional blood vessels. The use of three-dimensional (3D) bioprinting technologies has the potential to improve the classical tissue engineering approach because these will allow the generation of scaffolds with high spatial control of endothelial cell allocation. This study focuses on a side by side comparison of popular commercially available bioprinting hydrogels (Matrigel, fibrin, collagen, gelatin, agarose, Pluronic F-127, alginate, and alginate/gelatin) in the context of their physicochemical parameters, their swelling/degradation characteristics, their biological effects on vasculogenesis-related EC parameters and their printability. The aim of this study was to identify the most suitable hydrogel or hydrogel combination for inkjet printing of ECs to build prevascularized tissue constructs. Most tested hydrogels displayed physicochemical characteristics suitable for inkjet printing. However, Pluronic F-127 and the alginate/gelatin blend were rapidly degraded when incubated in cell culture medium. Agarose, Pluronic F-127, alginate and alginate/gelatin hydrogels turned out to be unsuitable for bioprinting of ECs because of their non-adherent properties and/or their incapability to support EC proliferation. Gelatin was able to support EC proliferation and viability but was unable to support endothelial cell sprouting. Our experiments revealed fibrin and collagen to be most suitable for bioprinting of ECs, because these hydrogels showed acceptable swelling/degradation characteristics, supported vasculogenesis-related EC parameters and showed good printability. Moreover, ECs in constructs of preformed spheroids survived the printing process and formed capillary-like cords. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 935-947, 2018.
Subject(s)
Bioprinting/methods , Human Umbilical Vein Endothelial Cells/cytology , Hydrogels/pharmacology , Animals , Cell Death/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Collagen/pharmacology , Elasticity , Fibrin/pharmacology , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Mice , Neovascularization, Physiologic/drug effects , Rats , Reproducibility of Results , Spheroids, Cellular/cytology , Spheroids, Cellular/drug effects , Surface Tension , ViscosityABSTRACT
Mesenchymal stem cells (MSCs) represent a very attractive cell source for tissue engineering applications aiming at the generation of artificial bone substitutes. The use of three-dimensional bioprinting technologies has the potential to improve the classical tissue engineering approach because bioprinting will allow the generation of hydrogel scaffolds with high spatial control of MSC allocation within the bioprinted construct. In this study, we have performed direct comparisons between commercially available hydrogels in the context of their cytocompatibility toward MSCs and their physicochemical parameters with the aim to identify the most suitable hydrogel for drop-on-demand (DoD) printing of MSCs. In this context, we examined matrigel, fibrin, collagen, gelatin, and gelatin/alginate at various hydrogel concentrations. Matrigel, fibrin, collagen, and gelatin were able to support cell viability, but the latter showed a limited potential to promote MSC proliferation. We concentrated our study on fibrin and collagen hydrogels and investigated the effect of hydroxyapatite (HA) inclusion. The inclusion of HA enhanced proliferation and osteogenic differentiation of MSCs and prevented degradation of fibrin in vitro. According to viscosity and storage moduli measurements, HA-blends displayed physicochemical characteristics suitable for DoD printing. In bioprinting experiments, we confirmed that fibrin and collagen and their respective HA-blends represent excellent hydrogels for DoD-based printing as evidenced by high survival rates of printed MSCs. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3231-3241, 2017.
Subject(s)
Biocompatible Materials/chemistry , Bioprinting , Hydrogel, Polyethylene Glycol Dimethacrylate/chemistry , Mesenchymal Stem Cells/cytology , Tissue Engineering , Tissue Scaffolds/chemistry , Alginates/chemistry , Bioprinting/methods , Cell Proliferation , Cell Survival , Cells, Cultured , Collagen/chemistry , Drug Combinations , Fibrin/chemistry , Gelatin/chemistry , Glucuronic Acid/chemistry , Hexuronic Acids/chemistry , Humans , Laminin/chemistry , Osteogenesis , Proteoglycans/chemistry , Tissue Engineering/methodsABSTRACT
Intratumoral genetic heterogeneity may impact disease outcome. Gold standard for dissecting clonal heterogeneity are single-cell analyses. Here, we present an efficient workflow based on an advanced Single-Cell Printer (SCP) device for the study of gene variants in single cancer cells. To allow for precise cell deposition into microwells the SCP was equipped with an automatic dispenser offset compensation, and the 384-microwell plates were electrostatically neutralized. The ejection efficiency was 99.7% for fluorescent beads (n = 2304) and 98.7% for human cells (U-2 OS or Kasumi-1 cancer cell line, acute myeloid leukemia [AML] patient; n = 150). Per fluorescence microscopy, 98.8% of beads were correctly delivered into the wells. A subset of single cells (n = 81) was subjected to whole genome amplification (WGA), which was successful in all cells. On empty droplets, a PCR on LINE1 retrotransposons yielded no product after WGA, verifying the absence of free-floating DNA in SCP-generated droplets. Representative gene variants identified in bulk specimens were sequenced in single-cell WGA DNA. In U-2 OS, 22 of 25 cells yielded results for both an SLC34A2 and TET2 mutation site, including cells harboring the SLC34A2 but not the TET2 mutation. In one cell, the TET2 mutation analysis was inconclusive due to allelic dropout, as assessed via polymorphisms located close to the mutation. Of Kasumi-1, 23 of 33 cells with data on both the KIT and TP53 mutation site harbored both mutations. In the AML patient, 21 of 23 cells were informative for a TP53 polymorphism; the identified alleles matched the loss of chromosome arm 17p. The advanced SCP allows efficient, precise and gentle isolation of individual cells for subsequent WGA and routine PCR/sequencing-based analyses of gene variants. This makes single-cell information readily accessible to a wide range of applications and can provide insights into clonal heterogeneity that were indeterminable solely by analyses of bulk specimens.